Candida antarctica lipase B catalyzed enantioselective acylation of pyrimidine acyclonucleoside

Abstract

The influence of solvent and acyl group donor on selectivity of the transesterification reaction of 1-[1′,3′-dihydroxy-2′-propoxymethyl]-5-methyluracil, a structural analogue of ganciclovir was examined. Lipase (EC 3.1.1.3) B from Candida antarctica (CALB) enabled desymmetrization of prochiral hydroxyl groups when 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF₆]) was used as a reaction medium. It was observed that CALB was up to 2.7–4 times more enantioselective in the ionic liquid [Bmim][PF₆] than in conventional organic solvents.     

Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoyl monoacylglycerol

Abstract

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was carried out by using several immobilized lipases in a solvent-free system. This reaction involves the initial formation of sn-2 docosahexaenyl monoacylglycerol. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp. (AQ) and Candida antarctica-fraction B (CALB) were immobilized by interfacial adsorption on a commercial hydrophobic support (a methacrylate resin containing octadecyl groups, Sepabeads C-18) and tested for glycerolysis of DHA ethyl ester. In certain cases (e.g. immobilized CALB), the transesterification reaction continues to the formation of triacylglycerol (80%) by using a very high excess of DHA ethyl ester ((115 mmols versus 1.24 mmols of glycerol and high temperatures (50?°C). However, the same biocatalyst working at lower temperatures, 37?°C, synthetizes a 90% of sn-2 monoacylglycerol even in the presence of that a high excess of DHA ethyl ester. Interestingly, immobilized RML derivative synthesizes a 98% of sn-2 monoacylglyceride (2-MG) in 15?min at 37?°C with a 4% of immobilized biocatalyst. These high activity and regioselectivity under very mild reaction conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid as well as for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate commercial markers, the formation of the 2-MG was confirmed.     

The influence of glycerol on the binding of methotrexate to Mycobacterium tuberculosis dihydrofolate reductase: a molecular modelling study

The enzyme, dihydrofolate reductase (DHFR), from Mycobacterium tuberculosis (mt-DHFR) is believed to be a potential drug target for the treatment of tuberculosis. The co-crystal structure of mt-DHFR bound with glycerol (GOL), NAPDH and methotrexate (MTX) reveals a GOL binding site on the enzyme. This GOL binding site could be very important for the design of novel, selective mt-DHFR inhibitors, because this binding site is absent on human DHFR (h-DHFR). We have performed molecular dynamic simulations and free energy calculations to evaluate the binding affinity of GOL and its free energy contribution to the binding of MTX to mt-DHFR. The results showed that GOL does not bind tightly to mt-DHFR. Although GOL itself contributed free energy on MTX binding to mt-DHFR, GOL also increased the flexibilities of MTX, so that MTX cannot maintain strong electronic interactions with ARG32 and ARG60, which caused the total binding free energy to decrease. These data suggest that GOL binding is weak and it could be expelled from the binding site, to allow inhibitors containing appropriate side chains to bind. This observation can be used to inform future drug design studies, especially those aimed at improving drug selectivity against h-DHFR.     

Effect of Solvents and Kinetic Parameters on Synthesis of Ethyl Propionate Catalysed by Poly (AAc-co-HPMA-cl-MBAm)-Matrix-Immobilized Lipase of Pseudomonas aeruginosa BTS-2.

Summary A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa BTS-2 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel network. The hydrogel showed approximately 95% binding efficiency for lipase (specific activity 1.96 U mg⁻¹). The immobilized enzyme achieved 65.1% conversion of ethanol and propionic acid (100 mM each) into ethyl propionate in n-nonane at 65 °C in 9 h. When alkane of C-chain length lower than n-nonane was used as the organic solvent, the conversion of ethanol and propionic acid into ethyl propionate decreased with a decrease in the log P value of alkanes. The immobilized lipase retained approximately 30% of its original catalytic activity after five cycles of reuse for esterification of ethanol and propionic acid into ethyl propionate at temperature 65 °C in 3 h. Addition of a molecular sieve (3 ?) to the reaction mixture enhanced the formation of ethyl propionate to 89.3%. Moreover, ethanol and propionic acid when taken a molar ratio of 3:1 further promoted the conversion rate to 94%. However, an increase in the molar ratio of propionic acid with respect to ethanol resulted in a decline of ethyl propionate synthesis.     

Modification Effects of Organic Solvents on Microenvironment of the Enzyme in Thermolysin-catalyzed Peptide Synthesis of N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine Methyl Ester

Thermolysin-catalyzed peptide synthesis using N-benzyloxycarbonyl)-l-phenylalanine (Z-Phe) and l-phenylalanine methyl ester (Phe-OMe) as substrates was done mainly in a water-organic one phase solvent system. The organic solvent content used was less than the saturation concentration in buffer. With organic solvents with high log P values, the enzymatic activity increased as the organic solvent content increased; but further increases in the organic solvent content decreased the enzymatic activity, showing an “organic activity” profile. On the other hand, with organic solvents of low log P values, the enzymatic reaction was inhibited even by the initial addition of organic solvents. When a correlation between maximum activities and logP values or Hildebrand solubility parameters was investigated, a linear correlation was obtained among the same category of organic solvents, but not between categories. This suggests that the direct effect of organic solvents on the microenvironment of the enzyme largely depends on the molecular structure of the solvents.     

Organic solvents for bioorganic synthesis

Summary The influence of solvents on enzymatic activity and stability was investigated. As a model reaction the -chymotrypsin-catalyzed esterification of N-acetyl-l-phenylalanine with ethanol was used. The enzyme was adsorbed on porous glass beads and used in various solvents. Small amounts of water were added to increase the enzymatic activity. These enzyme preparations obeyed. Michaelis-Menten kinetics. K _m,app decreased slightly with the log P value of the solvent while V _app increased markedly with the log P value. Log P values were also useful for generalizing the influence of solvents on enzyme stability. The enzyme preparations showed a markedly higher thermostability in dry solvents having log P values >0.7 than in less hydrophobic solvents.Also the operational stability was better in the more hydrophobic solvents. The amount of water added to the enzyme preparations greatly influenced the initial reaction rates. For some solvents optimal water contents were determined. The thermostability decreased with increasing water content.The observations are summarized in the conclusion that more hydrophobic solvents are preferable to less hydrophobic ones. The log P value gives a good guidance when selecting an organic solvent for enzymatic conversions.     

甘油影响红曲菌产色素的碳源选择性探究

红曲菌(Monascus spp.)是我国重要的药食同源微生物,红曲色素(Monascus pigments,MPs)是其主要次级代谢产物之一。有研究表明,甘油可促进红曲菌产MPs,但作用机制不明。以丛毛红曲菌(Monascus pilosus)MS-1为实验菌株,考察甘油与葡萄糖或蔗糖复合对红曲菌产MPs的影响。在不含碳源的合成培养基中,将甘油与葡萄糖或蔗糖复合,采用分光光度法和高效液相色谱法等分析MPs的产量和组分、生物量及发酵液pH。当甘油与葡萄糖复合,添加甘油后发酵液pH、生物量无显著变化(P0.05),总色价显著降低(P0.05)。当2 g/L或40 g/L甘油与蔗糖复合,发酵液pH显著降低而生物量及总色价显著增加(P0.05)。当40 g/L甘油与蔗糖复合时,总色价是仅以蔗糖为碳源时的16.5倍,且MPs同系物数量明显增多(P0.05)。在合成培养基条件下,甘油促进红曲菌产MPs具有碳源种类的选择性。该结果可为研究甘油影响红曲菌产MPs的作用机制提供参考,为甘油用于MPs生产提供依据。     

Effect of organic solvents on the activity and stability of an extracellular protease secreted by the haloalkaliphilic archaeon <Emphasis Type="Italic">Natrialba magadii</Emphasis>

The effect of various organic solvents on the activity and stability of an extracellular protease produced by the haloalkaliphilic archaeon Natrialba magadii was tested. This protease was active and stable in aqueous-organic solvent mixtures containing 1.5 M NaCl and glycerol, dimethylsulfoxide (DMSO), N,N-dimethyl formamide, propylenglycol, and dioxane. Among the solvents tested, DMSO, propylenglycol, and glycerol were effective in preserving enzyme stability in suboptimal NaCl concentrations. The stabilizing effect of DMSO on this haloalkaliphilic protease was more efficient at pH 8 than at pH 10, suggesting that DMSO may not substitute for salt to allow halophilic proteins to withstand the effect of high pH values. These results show that Nab. magadii extracellular protease is a solvent tolerant enzyme and suggest a potential application of this haloalkaliphilic protease in aqueous-organic solvent biocatalysis.     

Analysis of the Conformational Stability and Activity of Candida antarctica Lipase B in Organic Solvents: INSIGHT FROM MOLECULAR DYNAMICS AND QUANTUM MECHANICS/SIMULATIONS*

The conformational stability and activity of Candida antarctica lipase B (CALB) in the polar and nonpolar organic solvents were investigated by molecular dynamics and quantum mechanics/molecular mechanics simulations. The conformation change of CALB in the polar and nonpolar solvents was examined in two aspects: the overall conformation change of CALB and the conformation change of the active site. The simulation results show that the overall conformation of CALB is stable in the organic solvents. In the nonpolar solvents, the conformation of the active site keeps stable, whereas in the polar solvents, the solvent molecules reach into the active site and interact intensively with the active site. This interaction destroys the hydrogen bonding between Ser¹⁰⁵ and His²²⁴. In the solvents, the activation energy of CALB and that of the active site region were further simulated by quantum mechanics/molecular mechanics simulation. The results indicate that the conformation change in the region of active sites is the main factor that influences the activity of CALB.     

Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.     

Cation dependent O-methyltransferases from rice

Two lower molecular mass OMT genes (ROMT-15 and -17) were cloned from rice and expressed in Escherichia coli as glutathione S-transferase fusion proteins. ROMT-15 and -17 metabolized caffeoyl-CoA, flavones and flavonols containing two vicinal hydroxyl groups, although they exhibited different substrate specificities. The position of methylation in both luteolin and quercetin was determined to be the 3′ hydroxyl group and myricetin and tricetin were methylated not only at 3′ but also at 5′ hydroxyl groups. ROMT-15 and -17 are cation-dependent and mutation of the predicted metal binding sites resulted in the loss of the enzyme activity, indicating that the metal ion has a critical role in the enzymatic methylation.     

Lipase-catalyzed synthesis of glycerol carbonate from renewable glycerol and dimethyl carbonate through transesterification

Glycerol carbonate is a key multifunctional compound employed as solvent, additive, monomer, and chemical intermediate. Enzymatic synthesis of glycerol carbonate from renewable starting materials (glycerol and dimethyl carbonate) was successfully achieved by immobilized lipase from Candida antarctica (CALB, Novozym 435). Addition of molecular sieves as scavenger for the removal of methanol, which was generated from dimethyl carbonate during the reaction, accelerated a reaction rate. After the optimization, the equimolar use of glycerol and dimethyl carbonate in the Novozym 435-catalyzed reaction yielded a glycerol carbonate with almost quantitative yield. The resulting glycerol carbonate from 60 °C reaction has shown the low enantiomeric excess (13% ee) as configuration of (R)-enantiomer.     

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.     

Synthesis of propyl gallate by mycelium-bound tannase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in organic solvent

Aspergillus niger with mycelium-bound tannase activity was employed to investigate the synthesis of propyl gallate from gallic acid and 1-propanol in organic solvents. The effects of various organic solvents (log P: −1.0 to 6.6) on the enzymatic reactions showed that benzene (log P: 2.0) was the most suitable solvent. The water content and protonation state of mycelium-bound enzyme both had significant effects on the activity of tannase. The maximum molar conversion (65%) was achieved with 7.3% (v/v) 1-propanol and 5.56 mM gallic acid at stirring speeds of 200 rev/min, 40 °C in presence of anhydrous sodium sulfate and PEG-10,000. Enzyme specificity for the alcohol portion (C₁–C₈) of the ester showed that the optimum synthesis was observed with alcohols ranging from C₃ to C₅.     

Fructose-1,6-bisphosphatase from a cold-hardy insect: Control of cryoprotectant glycerol catabolism

Fructose 1,6-bisphosphatase (FBPase) from the larvae of the gall moth, Epiblema scudderiana, was purified to homogeneity with a final specific activity of 1.6 U/mg protein. The enzyme had a native molecular weight of 74.0 ± 6.5 kD and a subunit molecular weight of 37.6 ± 3.0 kD; the dimeric structure of the enzyme in this species is unusual. The pH optimum was 7.00 in imidazole buffer at 22°C and rose to 7.31 at 5°C. An Arrhenius plot of enzyme activity vs. temperature was linear with an activation energy of 91 ± 4.1 kJ/mol^?1. K_m values for FBPase decreased from 4.7 ± 0.34 μM at 22°C to 1.3 ± 0.05 μM at 5°C. No allosteric activators were identified, but the enzyme was inhibited by fructose 2,6-bisphosphate (F2,6P₂), AMP, ADP, dihydroxyacetonephosphate, glycerol, and KCI. Inhibition by AMP and F2,6P₂ increased at low temperature, and effects of these compounds may be key to preventing futile cycling of carbon at the FBPase/phosphofructokinase loci during the biosynthesis of glycerol cryoprotectant. Oppositely, glycerol clearance in the spring and reconversion into glycogen is promoted by interactions of temperature, inhibitors, and glycerol that promote FBPase activity: I₅₀ values for AMP and F2,6P₂ increase at 22°C (compared with 5°C), high glycerol levels override F2,6P₂ inhibition of the enzyme, and deinhibitors (ATP, citrate) partially reverse AMP inhibition of the enzyme. © 1995 Wiley-Liss, Inc.     

Optimization of the coating of octyl-CALB with ionic polymers to improve stability and decrease enzyme leakage

Abstract

Lipase B from Candida antarctica (CALB) immobilized on octyl-agarose (OC) was submitted to coating with polyethylenimine (PEI) and dextran sulfate (DS). Using lowly loaded enzyme preparations, the properties of OC-CALB preparations hardly improved, suggesting too large the distance between enzyme molecules. However, using OC-CALB preparations with maximum loading, CALB stability was greatly improved in different conditions after PEI coating. Moreover, the CALB release from the OC support in the presence of detergents, or during thermal or organic solvent inactivations was greatly reduced after this treatment (PEI plus DS coating). The results pointed that the main positive effect of this coating could be derived from the physical intermolecular crosslinking of the CALB molecules with the polymers that reduce the enzyme desorption from the support. The coating of OC-CALB-PEI with DS only produced a minimal improvement on enzyme performance. Even though the enzyme release was much more difficult after physical crosslinking, all enzyme molecules could be released from the OC support combining an ionic detergent (SDS), high buffer concentration, pH 3 and 45?°C, while using the OC-CALB just 2% SDS at pH 7 and 25?°C was enough to release all enzyme. The support could be reused several cycles. Thus, this strategy permitted to greatly reduce the enzyme desorption during operation and to improve enzyme stability while keeping the enzyme immobilization reversibility.     

Lipase-catalysed kinetic resolution of secondary alcohols with improved enantioselectivity in propylene carbonate

The lipase-catalysed kinetic resolution of secondary alcohols was studied using vinyl acetate as acyl donor in propylene carbonate. Propylene carbonate offers an environmentally friendly alternative in contrast to conventional solvents. Several different lipases were investigated, and Candida antarctica lipase B (CALB) exhibited better results for all the substrates. It was shown that the addition of non-reactive base triethylamine and silver oxide to the reaction mixture enhanced the reaction rate and enantioselectivity. With propylene carbonate as solvent, CALB could be recycled without significant activity or enantioselectivity losses.     

Catalytic characteristics of a sn-1(3) regioselective lipase from Cordyceps militaris

A total of 39 agricultural products were screened for natural sources of lipases with distinctive positional specificity. Based on this, Cordyceps militaris lipase (CML) was selected and subsequently purified by sequential chromatography involving anion-exchange, hydrophobic-interaction, and gel-permeation columns. As a result of the overall purification procedure, a remarkable increase in the specific activity of the CML (4.733 U/mg protein) was achieved, with a yield of 2.47% (purification fold of 94.54). The purified CML has a monomeric structure with a molecular mass of approximately 62 kDa. It was further identified as a putative extracellular lipase from C. militaris by the partial sequence analysis using ESI-Q-TOF MS. In a kinetic study of the CML-catalyzed hydrolysis, the values of V_max, K_m, and k_cat were determined to be 4.86 μmol·min⁻¹·mg⁻¹, 0.07 mM, and 0.29 min⁻¹, respectively. In particular, the relatively low K_m value indicated that CML has a high affinity for its substrate. With regard to positional specificity, CML selectively cleaved triolein at the sn-1 or 3 positions of glycerol backbone, releasing 1,2(2,3)-diolein as the major products. Therefore, CML can be considered a distinctive biocatalyst with sn-1(3) regioselectivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2744, 2019.     

The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L.

Developing cocoa cotyledons accumulate initially an unsaturated oil which is particularly rich in oleate and linoleate. However, as maturation proceeds, the characteristic high stearate levels appear in the storage triacylglycerols. In the early stages of maturation, tissue slices of developing cotyledons (105 days post anthesis, dpa) readily accumulate radioactivity from [¹⁴C]acetate into the diacylglycerols and label predominantly palmitate and oleate. In older tissues (130 dpa), by contrast, the triacylglycerols are extensively labelled and, at the same time, there is an increase in the percentage labelling of stearate. Thus, the synthesis of triacylglycerol and the production of stearate are co-ordinated during development. The relative labelling of the phospholipids (particularly phosphatidylcholine) was rather low at both stages of development which contrasts with oil seeds that accumulate a polyunsaturated oil (e.g. safflower). Microsomal membrane preparations from the developing cotyledons readily utilised an equimolar [¹⁴C]acyl-CoA substrate (consisting of palmitate, stearate and oleate) and glycerol 3-phosphate to form phosphatidate, diacylglycerol and triacylglycerol. Analysis of the [¹⁴C]acyl constituents at the sn-1 and sn-2 positions of phosphatidate and diacylglycerol revealed that the first acylase enzyme (glycerol 3-phosphate acyltransferase) selectively utilised palmitate over stearate and excluded oleate, whereas the second acylase (lysophosphatidate acyltransferase) was highly selective for the unsaturated acyl-CoA. On the other hand, the third acylase (diacylglycerol acyltransferase) exhibited an almost equal selectivity for palmitate and stearate. Thus, stearate is preferentially enriched at position sn-3 of triacylglycerol at 120–130 dpa because of the relatively higher selectivity of the diacylglycerol acyltransferase for this fatty acid compared with those of the other two acylation enzymes.Abbreviation dpa days post anthesis We are grateful to Drs. G. Pettipher (Cadbury-Schweppes, Reading, UK), M. End and P. Hadley (Department of Horticulture, University of Reading) for the supply of cocoa pods and to the Agricultural and Food Research Council for financial support. We also wish to thank Dr. S. Stymne (Swedish University of Agricultural Sciences, Uppsala, Sweden) for a generous gift of acyl-CoA substrates.